Mapping Protein–Ligand Interactions with Proteolytic Fragmentation, Hydrogen/Deuterium Exchange-Mass Spectrometry

Gallagher, Elyssia S.; Hudgens, Jeffrey W.

doi:10.1016/bs.mie.2015.08.010

Cited by 60 publications

(73 citation statements)

References 112 publications

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“…The numerous trajectories envisioned in the many-pathway model have been detected and characterized (18)(19)(20) and so can be considered on their merits rather than by abstract inference. Continued methods development (21,22) has enhanced the search for foldons and the investigation of their role in both equilibrium and kinetic folding (16,17). Advances in computational methods add a new vantage point (23)(24)(25)(26).…”

mentioning

confidence: 99%

The case for defined protein folding pathways

Englander

Mayne

2017

Proc. Natl. Acad. Sci. U.S.A.

124

143

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We consider the differences between the many-pathway protein folding model derived from theoretical energy landscape considerations and the defined-pathway model derived from experiment. A basic tenet of the energy landscape model is that proteins fold through many heterogeneous pathways by way of amino acid-level dynamics biased toward selecting native-like interactions. The many pathways imagined in the model are not observed in the structureformation stage of folding by experiments that would have found them, but they have now been detected and characterized for one protein in the initial prenucleation stage. Analysis presented here shows that these many microscopic trajectories are not distinct in any functionally significant way, and they have neither the structural information nor the biased energetics needed to select native vs. nonnative interactions during folding. The opposed defined-pathway model stems from experimental results that show that proteins are assemblies of small cooperative units called foldons and that a number of proteins fold in a reproducible pathway one foldon unit at a time. Thus, the same foldon interactions that encode the native structure of any given protein also naturally encode its particular foldon-based folding pathway, and they collectively sum to produce the energy bias toward native interactions that is necessary for efficient folding. Available information suggests that quantized native structure and stepwise folding coevolved in ancient repeat proteins and were retained as a functional pair due to their utility for solving the difficult protein folding problem.protein folding | foldons | energy landscape theory P rotein folding is among the most important reactions in all of biology. However, 50 y after C. B. Anfinsen showed that proteins can fold spontaneously without outside help (1, 2), and despite the intensive work of thousands of researchers leading to more than five publications per day in the current literature, there is still no general agreement on the most primary questions (3-5). How do proteins fold? Why do they fold in that way? How is the course of folding encoded in a 1D amino acid sequence? These questions have fundamental significance for protein science and its numerous applications. Over the years these questions have generated a large literature leading to different models for the folding process.The "new view" folding model, derived from hypothetical energy landscape and statistical mechanical considerations (6-12), proposes that folding proteins navigate to their native state through very many independent pathways. The many trajectories in Fig. 1A imply an unfolded protein conformationally searching for its lowestenergy native state by way of dynamic bond rotations in a way that is guided by its energy landscape, as for any chemical reaction. For effective performance, folding proteins must "know" how to select native as opposed to nonnative interactions. This information is said to be contained in the shape of the energy landscape, but how it is ...

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mentioning

confidence: 99%

The case for defined protein folding pathways

Englander

Mayne

2017

Proc. Natl. Acad. Sci. U.S.A.

124

143

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“…In principle, DXMS measures solvent accessibility of amide protons by quantifying their exchange for deuterium when a protein is incubated in D 2 O. Although there are other potential explanations for hydrogen/deuterium exchange (i.e., changes in hydrogen bonding, oligomerization, distal changes in protein conformation, and interaction with ligand), DXMS can be informative about local changes in protein conformation when coupled with other structural information (Cao et al, 2013; Gallagher & Hudgens, 2016; Percy et al, 2012). This approach has led to mapping of conformational changes in the group IVA phospholipase A 2 (cPLA 2 α) and group IA phospholipase A 2 upon binding to membranes, substrates, inhibitors, and divalent cations (Burke et al, 2009, 2008; Cao et al, 2013; Hsu et al, 2008; Mouchlis et al, 2015; Mouchlis & Dennis, 2016).…”

Section: Methodsmentioning

confidence: 99%

“…For additional detail on the theory, implementation, and optimization of DXMS, the reader is referred to two excellent reviews (Gallagher & Hudgens, 2016; Percy et al, 2012). The optimized protocol for analysis of iPLA 2 β is described in detail later:…”

Section: Methodsmentioning

confidence: 99%

“…As exchange of amide protons is negligible at pH 2.5 (Gallagher & Hudgens, 2016), the hydrogen/deuterium exchange reaction is quenched through addition of 100 μL of 0.8% formic acid, 2 M guanidine hydrochloride. This solution also denatures the protein, in preparation for protease digestion in the next phase.…”

Section: Methodsmentioning

confidence: 99%

“…Given its acid pH optimum, pepsin is among the most commonly used proteases to generate peptides for DXMS analyses (Gallagher & Hudgens, 2016; Percy et al, 2012). Porcine pepsin (Sigma, cat # P6887) is immobilized at 30 mg/mL on an Upchurch Scientific guard column (66 μL bed volume; cat # C.130B) packed with Poros 20 AL-activated affinity medium (ThermoFisher, cat # 1602802).…”

Section: Methodsmentioning

confidence: 99%

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Analyses of Calcium-Independent Phospholipase A2beta (iPLA2β) in Biological Systems

Barbour

Ramanadham

2017

Methods in Enzymology

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The Ca2+-independent phospholipases A2 (iPLA2s) are part of a diverse family of PLA2s, manifest activity in the absence of Ca2+, are ubiquitous, and participate in a variety of biological processes. Among the iPLA2s, the cytosolic iPLA2β has received considerable attention and ongoing studies from various laboratories suggest that dysregulation of iPLA2β can have a profound impact on the onset and/or progression of many diseases (e.g., cardiovascular, neurological, metabolic, autoimmune). Therefore, appropriate approaches are warranted to gain a better understanding of the role of iPLA2β in vivo and its contribution to pathophysiology. Given that iPLA2β is very labile, its basal expression is low in a number of cell systems, and that crystal structure of iPLA2β is not yet available, careful and efficient protocols are needed to appropriately assess iPLA2β biochemistry, dynamics, and membrane association. Here, step-by-step details are provided to (a) measure iPLA2β-specific activity in cell lines or tissue preparations (using a simple radiolabel-based assay) and assess the impact of stimuli and inhibitors on resting- and disease-state iPLA2β activity, (b) purify the iPLA2β to near homogeneity (via sequential chromatography) from cell line or tissue preparations, enabling concentration of the enzyme for subsequent analyses (e.g., proteomics), and (c) employ hydrogen/deuterium exchange mass spectrometry analyses to probe both the structure of iPLA2β and dynamics of its association with the membranes, substrates, and inhibitors.

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Metal adduction in mass spectrometric analyses of carbohydrates and glycoconjugates

Gass

Quintero

Hatvany

et al. 2022

Mass Spectrometry Reviews

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Glycans, carbohydrates, and glycoconjugates are involved in many crucial biological processes, such as disease development, immune responses, and cell–cell recognition. Glycans and carbohydrates are known for the large number of isomeric features associated with their structures, making analysis challenging compared with other biomolecules. Mass spectrometry has become the primary method of structural characterization for carbohydrates, glycans, and glycoconjugates. Metal adduction is especially important for the mass spectrometric analysis of carbohydrates and glycans. Metal‐ion adduction to carbohydrates and glycoconjugates affects ion formation and the three‐dimensional, gas‐phase structures. Herein, we discuss how metal‐ion adduction impacts ionization, ion mobility, ion activation and dissociation, and hydrogen/deuterium exchange for carbohydrates and glycoconjugates. We also compare the use of different metals for these various techniques and highlight the value in using metals as charge carriers for these analyses. Finally, we provide recommendations for selecting a metal for analysis of carbohydrate adducts and describe areas for continued research.

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Mapping Protein–Ligand Interactions with Proteolytic Fragmentation, Hydrogen/Deuterium Exchange-Mass Spectrometry

Cited by 60 publications

References 112 publications

The case for defined protein folding pathways

The case for defined protein folding pathways

Analyses of Calcium-Independent Phospholipase A2beta (iPLA2β) in Biological Systems

Metal adduction in mass spectrometric analyses of carbohydrates and glycoconjugates

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